We have developed a system for the isolation of mammalian origins of DNA replication--particularly those activated at the beginning of DNA synthesis. The method entails synchronizing cells with the Alpha polymerase inhibitor, aphidocolin; releasing the cells for 1 minute; isolating nuclei and continuing DNA synthesis with mercurated nucleotides; freeing the nascent strands by branch migration; purifying the nascent strands by sucrose gradient centrifugation and affinity chromatography on mercaptan columns; tailing or restriction digesting and cloning. The first clones to be isolated are now being sequenced. In other experiments we have generated deletions and substitutions of the 27-base-pair palindromic sequence at the SV40 replication origin. From these molecules, heteroduplex molecules have been formed which obligatorily exist as cruciform structures. A number of properties of these molecules are under investigation including: (1) their ability to act as templates for RNA synthesis; (2) their ability to cause nucleosome phasing; (3) their ability to serve as primers for mammalian polymerase; and (4) their ability to bind the SV40 T antigen.